PROJECT SUMMARY Melanoma is responsible for over 80% of deaths from skin cancer1. The past decade of research has identified oncogenic drivers of melanoma?notably BRAF and NRAS?and developed several effective small- molecule inhibitors such as BRAF and MEK inhibitors2. However, the duration of response to these targeted therapies is relatively short-lived due to the emergence of drug resistance against single agents4. Combination therapies targeting multiple pathways will probably be required for long-term success. Thus, there is a need to discover new therapeutic targets for melanoma. Previous research in the Morrison laboratory showed that cardiac glycosides combine with MEK inhibitor to reduce tumor growth in patient-derived melanoma xenografts and extend xenografted mouse survival5. The combination of digitoxin (a cardiac glycoside) plus a MAPK pathway inhibitor disrupts intracellular ion homeostasis, acidifies the cytoplasm, dysregulates mitochondrial Ca2+ levels, and induces cell death in human melanoma cells as a consequence of combinatorial effects on ion transporters5. Based on these preclinical data, a clinical trial of digoxin and trametinib (a MEK inhibitor) was initiated in advanced refractory BRAF wild-type melanomas. Approximately 25% of patients have exhibited significant responses to this drug combination, greater than the 10% response rate expected with trametinib alone18. These data raise the question of whether inhibition of other ion transporters can yield even greater therapeutic responses. Preliminary data suggest that human melanoma cells also preferentially depend upon L-type Ca2+ channels. Data generated by the lab demonstrated that CACNA1D, which encodes the ?1 pore-forming subunit of the L-type Ca2+ channel, is overexpressed in melanomas compared to normal melanocytes and is recurrently mutated in melanoma. Multiple Ca2+ channel inhibitors were also preferentially toxic against melanoma cells compared to normal cells in cell culture and were effective in reducing tumor growth in patient- derived melanoma xenografts. I hypothesize that melanoma cells preferentially require CACNA1D such that L- type Ca2+ channel inhibition can promote tumor regression. The primary goals of this research are to 1) determine if CACNA1D is necessary for melanoma tumor growth, 2) determine whether mutant CACNA1D is sufficient to drive melanoma initiation and/or progression, and 3) investigate whether CACNA1D regulates ion gradient homeostasis in melanoma cells. The goals will be accomplished by using a combination of genetic, pharmacological, imaging, and biochemical techniques. The work described in this proposal may ultimately be used to develop new targeted therapies against melanoma.